Elite Event A2407-12 and Methods and Kits for Identifying Such Event in Biological Samples

ABSTRACT

Tools are provided which allow rapid and unequivocal identification elite event A2704-12 in biological samples.

FIELD OF THE INVENTION

This invention pertains to methods and kits for identifying inbiological samples the presence of plant material comprisingspecifically transformation event A2704-12, as well as transgenicsoybean plants, plant material and seeds containing such event. Thesoybean plants of the invention combine the herbicide tolerant phenotypewith an agronomic performance, genetic stability and adaptability todifferent genetic backgrounds equivalent to the non-transformed soybeanline in the absence of weed pressure.

BACKGROUND OF THE INVENTION

The phenotypic expression of a transgene in a plant is determined bothby the structure of the gene itself and by its location in the plantgenome. At the same time the presence of the transgene (in a foreignDNA) at different locations in the genome will influence the overallphenotype of the plant in different ways. The agronomically orindustrially successful introduction of a commercially interesting traitin a plant by genetic manipulation can be a lengthy procedure dependenton different factors. The actual transformation and regeneration ofgenetically transformed plants are only the first in a series ofselection steps, which include extensive genetic characterization,breeding, and evaluation in field trials, eventually leading to theselection of an elite event.

The unequivocal identification of an elite event is becomingincreasingly important in view of discussions on Novel Food/Feed,segregation of GMO and non-GMO products and the identification ofproprietary material. Ideally, such identification method is both quickand simple, without the need for an extensive laboratory set-up.Furthermore, the method should provide results that allow unequivocaldetermination of the elite event without expert interpretation, butwhich hold up under expert scrutiny if necessary.

A2704-12 was selected as an elite event in the development of soybean(Glycine max L.) resistant to the herbicide Liberty®, by transformationof soybean with a plasmid comprising the synthetic pat gene encodingtolerance to phosphinothricin and may be commercially sold as LibertyLink® soybean. The tools for use in simple and unequivocal methods foridentification elite event A2704-12 in biological samples are describedherein.

SUMMARY OF THE INVENTION

The present invention relates to methods for identifying elite eventA2704-12 in biological samples, which methods are based on primers orprobes which specifically recognize the 5′ and/or 3′ flanking sequenceof A2704-12.

More specifically, the invention relates to a method comprising ofamplifying a sequence of a nucleic acid present in biological samples,using a polymerase chain reaction with at least two primers, one ofwhich recognizes the 5′ or 3′ flanking region of A2704-12, the otherwhich recognizes a sequence within the foreign DNA, preferably to obtaina DNA fragment of between 100 and 500 bp. The primers may recognize asequence within the 5′ flanking region of A2704-12 (SEQ ID No. 1, fromposition 1 to position 209) or within the 3′ flanking region of A2704-12(complement of SEQ ID No 2 from position 569 to position 1000) and asequence within the foreign DNA (complement of SEQ ID No 1 from position210 to 720 or SEQ ID No 2 from position 1 to position 568),respectively. The primer recognizing the 5′flanking region may comprisethe nucleotide sequence of SEQ ID No. 4 and the primer recognizing asequence within the foreign DNA may comprise the nucleotide sequence ofSEQ ID No. 8 described herein.

The present invention more specifically relates to a method foridentifying elite event A2704-12 in biological samples, which methodcomprises amplifying a sequence of a nucleic acid present in abiological sample, using a polymerase chain reaction with two primershaving the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 8respectively, to obtain a DNA fragment of about 185 bp.

The present invention further relates to the specific flanking sequencesof A2704-12 described herein, which can be used to develop specificidentification methods for A2704-12 in biological samples. Moreparticularly, the invention relates to the 5′ and or 3′ flanking regionsof A2704-12 which can be used for the development of specific primersand probes as further described herein. The invention further relates toidentification methods for the presence of A2704-12 in biologicalsamples based on the use of such specific primers or probes. Primers mayconsist of a nucleotide sequence of 17 to about 200 consecutivenucleotides selected from the nucleotide sequence of SEQ ID No 1 fromnucleotide 1 to nucleotide 209 or the complement of the nucleotidesequence of SEQ ID 2 from nucleotide 569 to nucleotide 1000) combinedwith primers consisting of a nucleotide sequence of 17 to about 200consecutive nucleotides selected from the complement of the nucleotidesequence of SEQ ID No 1 from nucleotide 210 to nucleotide 720 or thenucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 569.Primers may also comprise these nucleotide sequences located at theirextreme 3′ end, and further comprise unrelated sequences or sequencesderived from the mentioned nucleotide sequences, but comprisingmismatches.

The invention further relates to kits for identifying elite eventA2704-12 in biological samples, said kits comprising at least one primeror probe which specifically recognizes the 5′ or 3′ flanking region ofA2704-12.

The kit of the invention may comprise, in addition to a primer whichspecifically recognizes the 5′ or 3′ flanking region of A2704-12, asecond primer which specifically recognizes a sequence within theforeign DNA of A2704-12, for use in a PCR identification protocol.Preferably, the kit of the invention comprises two specific primers, oneof which recognizes a sequence within the 5′ flanking region ofA2704-12, and the other which recognizes a sequence within the foreignDNA. Especially The primer recognizing the 5′flanking region maycomprises the nucleotide sequence of SEQ ID No. 4 and the primerrecognizing the transgene may comprises the nucleotide sequence of SEQID No. 8 or any other primer as described herein.

The invention further relates to a kit for identifying elite eventA2704-12 in biological samples, said kit comprising the PCR primershaving the nucleotide sequence of SEQ ID No. 4 and SEQ ID No. 8 for usein the A2704-12 PCR identification protocol described herein.

The invention also relates to a kit for identifying elite event A2704-12in biological samples, which kit comprises a specific probe having asequence which corresponds (or is complementary to) a sequence havingbetween 80% and 100% sequence identity with a specific region ofA2704-12. Preferably the sequence of the probe corresponds to a specificregion comprising part of the 5′ or 3′ flanking region of A2704-12. Mostpreferably the specific probe has (or is complementary to) a sequencehaving between 80% and 100% sequence identity to the sequence betweennucleotide 160 and 260 of SEQ ID No. 1 or the sequence betweennucleotide 520 and 620 of SEQ ID No 2.

The methods and kits encompassed by the present invention can be usedfor different purposes such as, but not limited to the following: toidentify the presence or absence of A2704-12 in plants, plant materialor in products such as, but not limited to food or feed products (freshor processed) comprising or derived from plant material; additionally oralternatively, the methods and kits of the present invention can be usedto identify transgenic plant material for purposes of segregationbetween transgenic and non-transgenic material; additionally oralternatively, the methods and kits of the present invention can be usedto determine the quality (i.e. percentage pure material) of plantmaterial comprising A2704-12.

The invention further relates to the 5′ and/or 3′ flanking regions ofA2704-12 as well as to the specific primers and probes developed fromthe 5′ and/or 3′ flanking sequences of A2704-12.

The invention also relates to soybean plants, parts thereof, cells,seeds and progeny plants comprising elite event A2704-12. Such plants,parts thereof, cells, seeds and progeny plants can be identified usingthe methods as herein described.

DETAILED DESCRIPTION

The incorporation of a recombinant DNA molecule in the plant genometypically results from transformation of a cell or tissue (or fromanother genetic manipulation). The particular site of incorporation iseither due to “random” integration or is at a predetermined location (ifa process of targeted integration is used).

The DNA introduced into the plant genome as a result of transformationof a plant cell or tissue with a recombinant DNA or “transforming DNA”,and originating from such transforming DNA is hereinafter referred to as“foreign DNA” comprising one or more “transgenes”. “Plant DNA” in thecontext of the present invention will refer to DNA originating from theplant which is transformed. Plant DNA will usually be found in the samegenetic locus in the corresponding wild-type plant. The foreign DNA canbe characterized by the location and the configuration at the site ofincorporation of the recombinant DNA molecule in the plant genome. Thesite in the plant genome where a recombinant DNA has been inserted isalso referred to as the “insertion site” or “target site”. Insertion ofthe recombinant DNA into the plant genome can be associated with adeletion of plant DNA, referred to as “target site deletion”. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20 bp, preferably at least 50 bp, and up to 5000 bpof the plant genome which is located either immediately upstream of andcontiguous with or immediately downstream of and contiguous with theforeign DNA. Transformation procedures leading to random integration ofthe foreign DNA will result in transformants with different flankingregions, which are characteristic and unique for each transformant. Whenthe recombinant DNA is introduced into a plant through traditionalcrossing, its insertion site in the plant genome, or its flankingregions will generally not be changed. An “insertion region” as usedherein refers to the region corresponding to the region of at least 40bp, preferably at least 100 bp, and up to 10000 bp, encompassed by thesequence which comprises the upstream and/or the downstream flankingregion of a foreign DNA in the plant genome. Taking into considerationminor differences due to mutations within a species, an insertion regionwill retain, upon crossing into a plant of the same species, at least85%, preferably 90%, more preferably 95%, and most preferably 100%sequence identity with the sequence comprising the upstream anddownstream flanking regions of the foreign DNA in the plant originallyobtained from transformation.

An event is defined as a (artificial) genetic locus that, as a result ofgenetic engineering, carries a transgene comprising at least one copy ofa gene of interest. The typical allelic states of an event are thepresence or absence of the foreign DNA. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. At the molecularlevel, an event can be characterized by the restriction map (e.g. asdetermined by Southern blotting), by the upstream and/or downstreamflanking sequences of the transgene, the location of molecular markersand/or the molecular configuration of the transgene. Usuallytransformation of a plant with a transforming DNA comprising at leastone gene of interest leads to a multitude of events, each of which isunique.

An elite event, as used herein, is an event which is selected from agroup of events, obtained by transformation with the same transformingDNA or by back-crossing with plants obtained by such transformation,based on the expression and stability of the transgene(s) and itscompatibility with optimal agronomic characteristics of the plantcomprising it. Thus the criteria for elite event selection are one ormore, preferably two or more, advantageously all of the following:

a) That the presence of the foreign DNA does not compromise otherdesired characteristics of the plant, such as those relating toagronomic performance or commercial value;

b) That the event is characterized by a well defined molecularconfiguration which is stably inherited and for which appropriate toolsfor identity control can be developed;

c) That the gene(s) of interest show(s) a correct, appropriate andstable spatial and temporal phenotypic expression, both in heterozygous(or hemizygous) and homozygous condition of the event, at a commerciallyacceptable level in a range of environmental conditions in which theplants carrying the event are likely to be exposed in normal agronomicuse.

It is preferred that the foreign DNA is associated with a position inthe plant genome that allows easy introgression into desired commercialgenetic backgrounds.

The status of an event as an elite event is confirmed by introgressionof the elite event in different relevant genetic backgrounds andobserving compliance with one, two or all of the criteria e.g. a), b)and c) above.

An “elite event” thus refers to a genetic locus comprising a foreignDNA, which answers to the above-described criteria. A plant, plantmaterial or progeny such as seeds can comprise one or more elite eventsin its genome.

The tools developed to identify an elite event or the plant, plantmaterial comprising an elite event, or products which comprise plantmaterial comprising the elite event are based on the specific genomiccharacteristics of the elite event, such as, a specific restriction mapof the genomic region comprising the foreign DNA, molecular markers orthe sequence of the flanking region(s) of the foreign DNA.

Once one or both of the flanking regions of the foreign DNA have beensequenced, primers and probes can be developed which specificallyrecognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of asample by way of a molecular biological technique. For instance a PCRmethod can be developed to identify the elite event in biologicalsamples (such as samples of plants, plant material or productscomprising plant material). Such a PCR is based on at least two specific“primers” one recognizing a sequence within the 5′ or 3′ flanking regionof the elite event and the other recognizing a sequence within theforeign DNA. The primers preferably have a sequence of between 15 and 35nucleotides which under optimized PCR conditions “specificallyrecognize” a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event respectively, so that aspecific fragment (“integration fragment” or discriminating amplicon) isamplified from a nucleic acid sample comprising the elite event. Thismeans that only the targeted integration fragment, and no other sequencein the plant genome or foreign DNA, is amplified under optimized PCRconditions.

PCR primers suitable for the invention may be the following:

-   -   oligonucleotides ranging in length from 17 nt to about 210 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides selected from        the 5′ flanking sequence (SEQ ID No 1 from nucleotide 1 to        nucleotide 209) at their 3′ end (primers recognizing 5′ flanking        sequences); or    -   oligonucleotides ranging in length from 17 nt to about 450 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the 3′ flanking sequence (complement of SEQ ID No 2 from        nucleotide 569 to nucleotide 1000) at their 3′ end (primers        recognizing 3′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 510 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (complement of SEQ ID No 1 from        nucleotide 210 to nucleotide 720) at their 3′ end (primers        recognizing foreign DNA) or    -   oligonucleotides ranging in length from 17 nt to about 570 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (SEQ ID No 2 from nucleotide 1 to        nucleotide 569)

The primers may of course be longer than the mentioned 17 consecutivenucleotides, and may e.g. be 20, 21, 30, 35, 50, 75, 100, 150, 200 ntlong or even longer. The primers may entirely consist of nucleotidesequence selected from the mentioned nucleotide sequences of flankingsequences and foreign DNA sequences. However, the nucleotide sequence ofthe primers at their 5′ end (i.e. outside of the 3′-located 17consecutive nucleotides) is less critical. Thus, the 5′ sequence of theprimers may consist of a nucleotide sequence selected from the flankingsequences or foreign DNA, as appropriate, but may contain several (e.g.1, 2, 5, 10 mismatches). The 5′ sequence of the primers may evenentirely consist of a nucleotide sequence unrelated to the flankingsequences or foreign DNA, such as e.g. a nucleotide sequencerepresenting restriction enzyme recognition sites. Such unrelatedsequences or flanking DNA sequences with mismatches should preferably benot longer than 100, more preferably not longer than 50 or even 25nucleotides.

Moreover, suitable primers may comprise or consist of a nucleotidesequence at their 3′ end spanning the joining region between the plantDNA derived sequences and the foreign DNA sequences (located atnucleotides 209-210 in SEQ ID No 1 and nucleotides 568-569 in SEQ ID No2) provided the mentioned 3′-located 17 consecutive nucleotides are notderived exclusively from either the foreign DNA or plant-derivedsequences in SEQ ID No 1 or 2.

Thus, PCR primers suitable for the invention may also be the following:

-   -   oligonucleotides ranging in length from 17 nt to about 210 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides selected SEQ        ID No 1 from nucleotide 1 to nucleotide 215) at their 3′ end; or    -   oligonucleotides ranging in length from 17 nt to about 450 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the complement of SEQ ID No 2 from nucleotide 554 to        nucleotide 1000) at their 3′ end; or    -   oligonucleotides ranging in length from 17 nt to about 510 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        complement of SEQ ID No 1 from nucleotide 195 to nucleotide 720)        at their 3′ end or    -   oligonucleotides ranging in length from 17 nt to about 570 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from SEQ ID No 2        from nucleotide 1 to nucleotide 584)

It will also be immediately clear to the skilled artisan that properlyselected PCR primer pairs should also not comprise sequencescomplementary to each other.

For the purpose of the invention, the “complement of a nucleotidesequence represented in SEQ ID No: X” is the nucleotide sequence whichcan be derived from the represented nucleotide sequence by replacing thenucleotides through their complementary nucleotide according toChargaff's rules (A

T; G

C) and reading the sequence in the 5′ to 3′ direction, i.e in oppositedirection of the represented nucleotide sequence.

Examples of suitable primers are the oligonucleotide sequences of SEQ IDNo 3, SEQ ID No 4, SEQ ID No 5 (5′ flanking sequence recognizingprimers) SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No10, SEQ ID No 11 (foreign DNA recognizing primers for use with the 5′flanking sequence recognizing primers) SEQ ID No 12, SEQ ID No 13, SEQID No 14, SEQ ID No 15 (foreign DNA recognizing primers for use with the3′ flanking sequence recognizing primers) SEQ ID No 16, SEQ ID No 17,SEQ ID No 18 or SEQ ID No 19 (3′ flanking sequence recognizing primers).

Other examples of suitable oligonucleotide primers comprise at their 3′end the following sequences or consist of such sequences:

a. 5′ flanking sequence recognizing primers:

-   -   the nucleotide sequence of SEQ ID No 1 from nucleotide 23 to        nucleotide 42    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 68 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 69 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 69 to        nucleotide 88    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 134 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 22 to        nucleotide 42    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 30 to        nucleotide 49    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 67 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 70 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 70 to        nucleotide 88    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 76 to        nucleotide 95    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 78 to        nucleotide 97    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 133 to        nucleotide 152    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 21 to        nucleotide 42    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 31 to        nucleotide 49    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 34 to        nucleotide 63    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 66 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 68 to        nucleotide 88    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 73 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 75 to        nucleotide 95    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 77 to        nucleotide 97    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 77 to        nucleotide 95    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 134 to        nucleotide 152    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 154 to        nucleotide 173    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 22 to        nucleotide 43    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 33 to        nucleotide 53    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 35 to        nucleotide 53    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 67 to        nucleotide 88    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 72 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 74 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 76 to        nucleotide 97    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 78 to        nucleotide 95    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 135 to        nucleotide 152    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 154 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 32 to        nucleotide 53    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 36 to        nucleotide 57    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 71 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 74 to        nucleotide 95    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 75 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 32 to        nucleotide 51    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 31 to        nucleotide 51    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 33 to        nucleotide 51    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 30 to        nucleotide 51    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 34 to        nucleotide 51    -   the nucleotide sequence of SEQ ID No 1 from nucleotide 205 to        nucleotide 226

b. foreign DNA sequence recognizing primers for use with 5′ flankingsequence recognizing primers:

-   -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 201 to nucleotide 220    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 220 to nucleotide 239    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 361 to nucleotide 380    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 366 to nucleotide 385    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 201 to nucleotide 219    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 220 to nucleotide 238    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 221 to nucleotide 239    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 358 to nucleotide 377    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 359 to nucleotide 378    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 361 to nucleotide 379    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 366 to nucleotide 384    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 368 to nucleotide 387    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 496 to nucleotide 515    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 656 to nucleotide 675    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 201 to nucleotide 218    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 220 to nucleotide 237    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 221 to nucleotide 238    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 220 to nucleotide 240    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 221 to nucleotide 240    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 251 to nucleotide 270    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 252 to nucleotide 271    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 253 to nucleotide 272    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 359 to nucleotide 377    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 361 to nucleotide 378    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 358 to nucleotide 378    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 359 to nucleotide 379    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 361 to nucleotide 382    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 366 to nucleotide 383    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 368 to nucleotide 386    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 366 to nucleotide 386    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 375 to nucleotide 393    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 375 to nucleotide 394    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 496 to nucleotide 514    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 498 to nucleotide 515    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 562 to nucleotide 581    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 608 to nucleotide 627    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 651 to nucleotide 670    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 655 to nucleotide 674    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 656 to nucleotide 674    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 656 to nucleotide 676    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 669 to nucleotide 678    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 252 to nucleotide 270    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 253 to nucleotide 271    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 251 to nucleotide 271    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 252 to nucleotide 272    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 267 to nucleotide 286    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 359 to nucleotide 376    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 358 to nucleotide 379    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 362 to nucleotide 379    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 359 to nucleotide 380    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 368 to nucleotide 385    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 368 to nucleotide 388    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 375 to nucleotide 392    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 376 to nucleotide 393    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 376 to nucleotide 394    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 376 to nucleotide 395    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 440 to nucleotide 459    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 442 to nucleotide 461    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 496 to nucleotide 513    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 556 to nucleotide 575    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 558 to nucleotide 577    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 561 to nucleotide 580    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 563 to nucleotide 582    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 562 to nucleotide 582    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 608 to nucleotide 628    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 637 to nucleotide 656    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 644 to nucleotide 663    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 651 to nucleotide 669    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 651 to nucleotide 671    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 656 to nucleotide 673    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 655 to nucleotide 675    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 659 to nucleotide 677    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 659 to nucleotide 679    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 201 to nucleotide 222    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 204 to nucleotide 225    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 223 to nucleotide 240    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 223 to nucleotide 241    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 220 to nucleotide 241    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 221 to nucleotide 241    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 253 to nucleotide 270    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 251 to nucleotide 272    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 252 to nucleotide 273    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 253 to nucleotide 274    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 268 to nucleotide 289    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 377 to nucleotide 394    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 375 to nucleotide 395    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 440 to nucleotide 458    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 442 to nucleotide 460    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 440 to nucleotide 460    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 442 to nucleotide 462    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 492 to nucleotide 513    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 503 to nucleotide 522    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 556 to nucleotide 574    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 556 to nucleotide 576    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 562 to nucleotide 579    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 563 to nucleotide 581    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 563 to nucleotide 583    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 562 to nucleotide 583    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 608 to nucleotide 625    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 608 to nucleotide 629    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 644 to nucleotide 662    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 651 to nucleotide 668    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 651 to nucleotide 672    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 655 to nucleotide 676    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 655 to nucleotide 677    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 659 to nucleotide 680    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 657 to nucleotide 688    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 223 to nucleotide 242    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 267 to nucleotide 288    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 368 to nucleotide 389    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 375 to nucleotide 396    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 376 to nucleotide 397    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 440 to nucleotide 457    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 442 to nucleotide 459    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 442 to nucleotide 463    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 534 lo to nucleotide 553    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 558 to nucleotide 575    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 556 to nucleotide 577    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 563 to nucleotide 580    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 563 to nucleotide 584    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 644 to nucleotide 661    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 645 to nucleotide 662    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 644 to nucleotide 665    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 645 to nucleotide 666    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 325 to nucleotide 342    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 503 to nucleotide 520    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 534 to nucleotide 554    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 221 to nucleotide 242    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 505 to nucleotide 522    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 534 to nucleotide 551    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 534 to nucleotide 555    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 536 to nucleotide 557    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 551 to nucleotide 570    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 551 to nucleotide 571    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 551 to nucleotide 572

c. foreign DNA sequence recognizing primers for use with 3′ flankingsequence recognizing primers:

-   -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 955 to nucleotide 974    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 955 to nucleotide 973    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 955 to nucleotide 972    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 958 to nucleotide 975    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 958 to nucleotide 977    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 917 to nucleotide 934    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 947 to nucleotide 968    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 951 to nucleotide 968    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 951 to nucleotide 972    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 958 to nucleotide 976    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 955 to nucleotide 976    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 958 to nucleotide 979

d. 3′ flanking sequence recognizing primers:

-   -   the nucleotide sequence of SEQ ID No 2 from nucleotide 151 to        nucleotide 170    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 152 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 6 to        nucleotide 25    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 148 to        nucleotide 167    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 151 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 152 to        nucleotide 170    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 153 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 5 to        nucleotide 25    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 7 to        nucleotide 25    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 67 to        nucleotide 86    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 89 to        nucleotide 108    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 134 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 147 to        nucleotide 167    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 150 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 153 to        nucleotide 170    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 154 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 168 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 169 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 171 to        nucleotide 190    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 197 to        nucleotide 216    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 236 to        nucleotide 255    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 280 to        nucleotide 299    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 4 to        nucleotide 25    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 8 to        nucleotide 25    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 63 to        nucleotide 82    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 66 to        nucleotide 86    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 68 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 90 to        nucleotide 108    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 93 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 94 to        nucleotide 113    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 96 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 101 to        nucleotide 120    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 134 to        nucleotide 154    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 146 to        nucleotide 167    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 150 to        nucleotide 167    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 170 to        nucleotide 190    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 172 to        nucleotide 190    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 186 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 189 to        nucleotide 208    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 190 to        nucleotide 209    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 191 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 195 to        nucleotide 214    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 196 to        nucleotide 216    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 196 to        nucleotide 214    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 198 to        nucleotide 216    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 199 to        nucleotide 216    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 208 to        nucleotide 227    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 234 to        nucleotide 253    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 235 to        nucleotide 255    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 279 to        nucleotide 299    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 281 to        nucleotide 299    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 285 to        nucleotide 304    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 296 to        nucleotide 315    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 396 to        nucleotide 415    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 64 to        nucleotide 82    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 65 to        nucleotide 86    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 67 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 75 to        nucleotide 92    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 91 to        nucleotide 108    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 92 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 93 to        nucleotide 113    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 94 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 95 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 95 to        nucleotide 113    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 97 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 100 to        nucleotide 120    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 132 to        nucleotide 153    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 133 to        nucleotide 154    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 163 to        nucleotide 182    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 165 to        nucleotide 184    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 167 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 169 to        nucleotide 190    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 173 to        nucleotide 190    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 187 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 189 to        nucleotide 209    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 190 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 190 to        nucleotide 208    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 191 to        nucleotide 209    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 192 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 194 to        nucleotide 214    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 197 to        nucleotide 214    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 233 to        nucleotide 253    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 234 to        nucleotide 255    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 235 to        nucleotide 253    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 282 to        nucleotide 299    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 295 to        nucleotide 315    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 297 to        nucleotide 315    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 397 to        nucleotide 415    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 65 to        nucleotide 82    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 66 to        nucleotide 87    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 75 to        nucleotide 96    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 91 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 92 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 94 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 95 to        nucleotide 112    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 98 to        nucleotide 115    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 99 to        nucleotide 120    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 162 to        nucleotide 182    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 164 to        nucleotide 182    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 164 to        nucleotide 184    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 164 to        nucleotide 184    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 184 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 187 to        nucleotide 208    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 188 to        nucleotide 205    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 188 to        nucleotide 209    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 189 to        nucleotide 210    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 191 to        nucleotide 208    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 192 to        nucleotide 209    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 193 to        nucleotide 214    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 205 to        nucleotide 212    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 232 to        nucleotide 253    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 236 to        nucleotide 253    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 242 to        nucleotide 261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 278 to        nucleotide 299    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 283 to        nucleotide 304    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 287 to        nucleotide 304    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 294 to        nucleotide 315    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 298 to        nucleotide 315    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 332 to        nucleotide 349    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 398 to        nucleotide 415    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 161 to        nucleotide 182    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 163 to        nucleotide 184    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 165 to        nucleotide 182    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 166 to        nucleotide 187    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 241 to        nucleotide 261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 243 to        nucleotide 261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 244 to        nucleotide 261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 240 to        nucleotide 261    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 126 to        nucleotide 145    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 208 to        nucleotide 225    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 124 to        nucleotide 145    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 75 to        nucleotide 94    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 231 to        nucleotide 250    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 243 to        nucleotide 262    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 230 to        nucleotide 250    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 232 to        nucleotide 250    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 242 to        nucleotide 262    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 244 to        nucleotide 262    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 229 to        nucleotide 250    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 241 to        nucleotide 262    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 245 to        nucleotide 262    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 287 to        nucleotide 306    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 288 to        nucleotide 306    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 230 to        nucleotide 247    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 285 to        nucleotide 306    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 289 to        nucleotide 306    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 282 to        nucleotide 303    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 288 to        nucleotide 307    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 287 to        nucleotide 307    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 289 to        nucleotide 307    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 286 to        nucleotide 307    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 290 to        nucleotide 307    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 229 to        nucleotide 248    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 230 to        nucleotide 248    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 227 to        nucleotide 248    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 231 to        nucleotide 248

As used herein, “the nucleotide sequence of SEQ ID No. Z from position Xto position Y” indicates the nucleotide sequence including bothnucleotide endpoints.

Preferably, the integration fragment has a length of between 50 and 500nucleotides, most preferably of between 100 and 350 nucleotides. Thespecific primers may have a sequence which is between 80 and 100%identical to a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event, respectively, provided themismatches still allow specific identification of the elite event withthese primers under optimized PCR conditions. The range of allowablemismatches however, can easily be determined experimentally and areknown to a person skilled in the art.

The following table exemplifies the sizes of expected DNA amplicons ( orintegration fragments) with selected pairs of PCR primers.

Length Primer 1 From position Primer 2 To position amplicon HCA148 12KVM174 225 213 HCA148 12 KVM177 253 241 HCA148 12 DPA024 316 304 HCA14812 MDB390 396 384 HCA148 12 HCA023 511 499 HCA148 12 DPA007 634 622DPA021 134 KVM174 225 91 DPA021 134 KVM177 253 119 DPA021 134 DPA024 316182 DPA021 134 MDB390 396 262 DPA021 134 HCA023 511 377 DPA021 134DPA007 634 500 KVM176 187 KVM174 225 38 KVM176 187 KVM177 253 66 KVM176187 DPA024 316 129 KVM176 187 MDB390 396 209 KVM176 187 HCA023 511 324KVM176 187 DPA007 634 447 YTP007 116 HCA074 628 512 YTP007 116 SMO017667 551 YTP007 116 SMO027 710 594 YTP007 116 SMO033 867 751 MDB452 187HCA074 628 441 MDB452 187 SMO017 667 480 MDB452 187 SMO027 710 523MDB452 187 SMO033 867 680 HCA014 398 HCA074 628 230 HCA014 398 SMO017667 269 HCA014 398 SMO027 710 312 HCA014 398 SMO033 867 469 MDB402 528HCA074 628 100 MDB402 528 SMO017 667 139 MDB402 528 SMO027 710 182MDB402 528 SMO033 867 339

Detection of integration fragments can occur in various ways e.g. viasize estimation after gel analysis. The integration fragments may alsobe directly sequenced. Other sequence specific methods for detection ofamplified DNA fragments are also known in the art.

As the sequence of the primers and their relative location in the genomeare unique for the elite event, amplification of the integrationfragment will occur only in biological samples comprising (the nucleicacid of) the elite event. Preferably when performing a PCR to identifythe presence of A2704-12 in unknown samples, a control is included of aset of primers with which a fragment within a “housekeeping gene” of theplant species of the event can be amplified. Housekeeping genes aregenes that are expressed in most cell types and which are concerned withbasic metabolic activities common to all cells. Preferably, the fragmentamplified from the housekeeping gene is a fragment which is larger thanthe amplified integration fragment. Depending on the samples to beanalyzed, other controls can be included.

Standard PCR protocols are described in the art, such as in “PCRApplications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999).The optimal conditions for the PCR, including the sequence of thespecific primers, is specified in a “PCR identification protocol” foreach elite event. It is however understood that a number of parametersin the PCR identification protocol may need to be adjusted to specificlaboratory conditions, and may be modified slightly to obtain similarresults. For instance, use of a different method for preparation of DNAmay require adjustment of, for instance, the amount of primers,polymerase and annealing conditions used. Similarly, the selection ofother primers may dictate other optimal conditions for the PCRidentification protocol. These adjustments will however be apparent to aperson skilled in the art, and are furthermore detailed in current PCRapplication manuals such as the one cited above.

Alternatively, specific primers can be used to amplify an integrationfragment that can be used as a “specific probe” for identifying A2704-12in biological samples. Contacting nucleic acid of a biological sample,with the probe, under conditions which allow hybridization of the probewith its corresponding fragment in the nucleic acid, results in theformation of a nucleic acid/probe hybrid. The formation of this hybridcan be detected (e.g. labeling of the nucleic acid or probe), wherebythe formation of this hybrid indicates the presence of A2704-12. Suchidentification methods based on hybridization with a specific probe(either on a solid phase carrier or in solution) have been described inthe art. The specific probe is preferably a sequence which, underoptimized conditions, hybridizes specifically to a region within the 5′or 3′ flanking region of the elite event and preferably also comprisingpart of the foreign DNA contiguous therewith (hereinafter referred to as“specific region”). Preferably, the specific probe comprises a sequenceof between 50 and 500 bp, preferably of 100 to 350 bp which is at least80%, preferably between 80 and 85%, more preferably between 85 and 90%,especially preferably between 90 and 95%, most preferably between 95%and 100% identical (or complementary) to the nucleotide sequence of aspecific region. Preferably, the specific probe will comprise a sequenceof about 15 to about 100 contiguous nucleotides identical (orcomplementary) to a specific region of the elite event.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method of the invention, more particularly, theidentification of the elite event A2704-12 in biological samples. Moreparticularly, a preferred embodiment of the kit of the inventioncomprises at least one or two specific primers, as described above.Optionally, the kit can further comprise any other reagent describedherein in the PCR identification protocol. Alternatively, according toanother embodiment of this invention, the kit can comprise a specificprobe, as described above, which specifically hybridizes with nucleicacid of biological samples to identify the presence of A2704-12 therein.Optionally, the kit can further comprise any other reagent (such as butnot limited to hybridizing buffer, label) for identification of A2704-12in biological samples, using the specific probe.

The kit of the invention can be used, and its components can bespecifically adjusted, for purposes of quality control (e.g., purity ofseed lots), detection of the elite event in plant material or materialcomprising or derived from plant material, such as but not limited tofood or feed products.

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983,Proc. Nat. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides,a word length of 4 nucleotides, and a gap penalty of 4.Computer-assisted analysis and interpretation of sequence data,including sequence alignment as described above, can, e.g., beconveniently performed using the programs of the Intelligenetics™ Suite(Intelligenetics Inc., CA) or the sequence analysis software package ofthe Genetics Computer Group (GCG, University of Wisconsin Biotechnologycenter).

Sequences are indicated as “essentially similar” when such sequenceshave a sequence identity of at least about 75%, particularly at leastabout 80%, more particularly at least about 85%, quite particularlyabout 90%, especially about 95%, more especially about 100%. It is clearthan when RNA sequences are said to be essentially similar or have acertain degree of sequence identity with DNA sequences, thymidine (T) inthe DNA sequence is considered equal to uracil (U) in the RNA sequence.

The term “primer” as used herein encompasses any nucleic acid that iscapable of priming the synthesis of a nascent nucleic acid in atemplate-dependent process, such as PCR. Typically, primers areoligonucleotides from 10 to 30 nucleotides, but longer sequences can beemployed. Primers may be provided in double-stranded form, though thesingle-stranded form is preferred. Probes can be used as primers, butare designed to bind to the target DNA or RNA and need not be used in anamplification process.

The term “recognizing” as used herein when referring to specificprimers, refers to the fact that the specific primers specificallyhybridize to a nucleic acid sequence in the elite event under theconditions set forth in the method (such as the conditions of the PCRidentification protocol), whereby the specificity is determined by thepresence of positive and negative controls.

The term “hybridizing” as used herein when referring to specific probes,refer to the fact that the probe binds to a specific region in thenucleic acid sequence of the elite event under standard stringencyconditions. Standard stringency conditions as used herein refers to theconditions for hybridization described herein or to the conventionalhybridizing conditions as described by Sambrook et al., 1989 (MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarbourLaboratory Press, NY) which for instance can comprise the followingsteps: 1) immobilizing plant genomic DNA fragments on a filter, 2)prehybridizing the filter for 1 to 2 hours at 42° C. in 50% formamide,5×SSPE, 2×Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68° C.in 6×SSC, 2×Denhardt's reagent and 0.1% SDS, 3) adding the hybridizationprobe which has been labeled, 4) incubating for 16 to 24 hours, 5)washing the filter for 20 min. at room temperature in 1×SSC, 0.1% SDS,6) washing the filter three times for 20 min. each at 68° C. in 0.2×SSC,0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at−70° C. with an intensifying screen.

As used in herein, a biological samples is a sample of a plant, plantmaterial or products comprising plant material. The term “plant” isintended to encompass soybean (Glycine max) plant tissues, at any stageof maturity, as well as any cells, tissues, or organs taken from orderived from any such plant, including without limitation, any seeds,leaves, stems, flowers, roots, single cells, gametes, cell cultures,tissue cultures or protoplasts. “Plant material”, as used herein refersto material which is obtained or derived from a plant. Productscomprising plant material relate to food, feed or other products whichare produced using plant material or can be contaminated by plantmaterial. It is understood that, in the context of the presentinvention, such biological samples are tested for the presence ofnucleic acids specific for A2704-12, implying the presence of nucleicacids in the samples. Thus the methods referred to herein foridentifying elite event A2704-12 in biological samples, relate to theidentification in biological samples of nucleic acids which comprise theelite event.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, reagents or componentsas referred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA sequence which is functionallyor structurally defined, may comprise additional DNA sequences, etc.

The present invention also relates to the development of an elite eventA2704-12 in soybean to the plants comprising this event, the progenyobtained from these plants and to the plant cells, or plant materialderived from this event. Plants comprising elite event A2704-12 wereobtained through as described in example 1.

Soybean plants or plant material comprising A2704-12 can be identifiedaccording to the PCR identification protocol described for A2704-12 inExample 2. Briefly, soybean genomic DNA present in the biological sampleis amplified by PCR using a primer which specifically recognizes asequence within the 5′ or 3′ flanking sequence of A2704-12 such as theprimer with the sequence of SEQ ID NO: 4, and a primer which recognizesa sequence in the foreign DNA, such as the primer with the sequence ofSEQ ID NO: 8. DNA primers which amplify part of an endogenous soybeansequence are used as positive control for the PCR amplification. If uponPCR amplification, the material yields a fragment of the expected size,the material contains plant material from a soybean plant harboringelite event A2704-12.

Plants harboring A2704-12 are characterized by their glufosinatetolerance, which in the context of the present invention includes thatplants are tolerant to the herbicide Liberty™. Tolerance to Liberty™ canbe tested in different ways. The leaf paint method as described herein,is most useful when discrimination between resistant and sensitiveplants is required, without killing the sensitive ones. Alternatively,tolerance can be tested by Liberty™ spray application. Spray treatmentsshould be made between the leaf stages V3 and V4 for best results.Tolerant plants are characterized by the fact that spraying of theplants with at least 200 grams active ingredient/hectare (g.a.i./ha),preferably 400 g.a.i./ha, and possibly up to 1600 g.a.i./ha (4× thenormal field rate), does not kill the plants. A broadcast applicationshould be applied at a rate of 28-34 oz Liberty™. It is best to apply ata volume of 20 gallons of water per acre using a flat fan type nozzlewhile being careful not to direct spray applications directly into thewhorl of the plants to avoid surfactant burn on the leaves. Theherbicide effect should appear within 48 hours and be clearly visiblewithin 5-7 days.

Plants harboring A2704-12 can further be characterized by the presencein their cells of phosphinothricin acetyl transferase as determined by aPAT assay (De Block et al, 1987).

Plants harboring A2704-12 are also characterized by having agronomicalcharacteristics that are comparable to commercially available varietiesof soybean in the US, in the absence of weed pressure and use ofLiberty™ for weed control. It has been observed that the presence of aforeign DNA in the insertion region of the soybean plant genomedescribed herein, confers particularly interesting phenotypic andmolecular characteristics to the plants comprising this event. Morespecifically, the presence of the foreign DNA in this particular regionin the genome of these plants, results in plants which display a stablephenotypic expression of the gene of interest without significantlycompromising any aspect of desired agronomic performance of the plants.

The following examples describe the identification of the development oftools for the identification of elite event A2704-12 in biologicalsamples.

Unless otherwise stated, all recombinant DNA techniques are carried outaccording to standard protocols as described in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al.(1994) Current Protocols in Molecular Biology, Current Protocols, USA.Standard materials and methods for plant molecular work are described inPlant Molecular Biology Labfax (1993) by R. D. D. Croy published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications,UK.

In the description and examples, reference is made to the followingsequences:

SEQ ID No. 1: nucleotide sequence comprising a 5′ flanking region ofA2704-12 SEQ ID No. 2: nucleotide sequence comprising a 3′ flankingregion of A2704-12 SEQ ID No. 3: primer HCA148 SEQ ID No. 4: primerDPA021 SEQ ID No. 5: primer KVM176 SEQ ID No. 6: primer KVM174 SEQ IDNo. 7: primer KVM177 SEQ ID No. 8: primer DPA024 SEQ ID No. 9: primerMDB390 SEQ ID No. 10: primer HCA023 SEQ ID No. 11: primer DPA007 SEQ IDNo. 12: primer YTP007 SEQ ID No. 13: primer MDB452 SEQ ID No. 14: primerHCA014 SEQ ID No. 15: primer MDB402 SEQ ID No. 16: primer HCA074 SEQ IDNo. 17: primer SMO017 SEQ ID No. 18: primer SMO027 SEQ ID No. 19: primerSMO033 SEQ ID No. 20: primer 1 for amplification of control fragment SEQID No. 21: primer 2 for amplification of control fragment

BRIEF DESCRIPTION OF THE DRAWINGS

The following Examples, not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figure, incorporated herein by reference, in which:

FIG. 1: Schematic representation of the relationship between the citednucleotide sequences and primers. black bar: foreign DNA; light bar: DNAof plant origin; the figures under the bars represent nucleotidepositions; (c) refers to complement of the indicated nucleotidesequence.

FIG. 2: PCR Identification protocol developed for A2704-12. Loadingsequence of the gel: Lane 1: DNA sample from soybean plants comprisingthe transgenic event A2704-12; lane 2: DNA sample from a transgenicsoybean plant not comprising elite event A2704-12; lane 3: control DNAsamples from wild-type soybean plants; lane 4: no template control; lane5: molecular weight marker.

EXAMPLES

1. Identification of the flanking regions of elite event A2704-12

Herbicide-resistant soybean was developed by transformation of soybeanwith a vector comprising the coding sequence of a pat gene encoding theenzyme phosphinothricin-acetyl-transferase, under the control of theconstitutive 35S promoter from Cauliflower Mosaic virus.

Elite event A2704-12 was selected based on an extensive selectionprocedure based on good expression and stability of the herbicideresistance gene and its compatibility with optimal agronomiccharacteristics.

The sequence of the regions flanking the foreign DNA in the A2704-12event was determined using the thermal asymmetric interlaced (TAIL-) PCRmethod described by Liu et al. (1995, Plant J. 8(3):457-463). Thismethod utilizes three nested primers in successive reactions togetherwith a shorter arbitrary degenerate primer so that the relativeamplification efficiencies of specific and non-specific products can bethermally controlled. The specific primers were selected for annealingto the border of the foreign DNA and based on their annealingconditions. A small amount (5 μl) of unpurified, secondary and tertiary,PCR products were analyzed on a 1% agarose gel. The tertiary PCR productwas used for preparative amplification, purified and sequenced on anautomated sequencer using the DyeDeoxy Terminator cycle kit.

1.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region obtained bythe TAIL-PCR method was completely sequenced (SEQ ID No. 1). Thesequence between nucleotide 1 and 209 corresponds to plant DNA, whilethe sequence between nucleotide 210 and 720 corresponds to foreign DNA.

1.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region obtained bythe TAIL-PCR method was completely sequenced (SEQ ID No. 2). Thesequence between nucleotide 1 and 568 corresponds to foreign DNA, whilethe sequence between nucleotide 569 and 1000 corresponds to plant DNA.

2. Development of a Polymerase Chain Reaction Identification Protocol2.1. Primers

Specific primers were developed which recognize sequences within theelite event. More particularly, a primer was developed which recognizesa sequence within the 5′ flanking region of A2704-12. A second primerwas then selected within the sequence of the foreign DNA so that theprimers span a sequence of about 183 nucleotides. The following primerswere found to give particularly clear and reproducible results in a PCRreaction on A2704-12 DNA:

DPA021: 5′-ggC.gTT.CgT.AgT.gAC.TgA.gg-3′ (SEQ ID No.: 4) (target: plantDNA) DPA024: 5′-gTT.TTA.CAA.CgT.gAC.Tgg-3′ (SEQ ID No.: 8) (target:insert DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive lo control. A positive result with theendogenous primer-pair demonstrates that there is ample DNA of adequatequality in the genomic DNA preparation for a PCR product to begenerated. The endogenous primers were selected to recognize ahousekeeping gene in Glycine max:

SOY01: (SEQ ID No.: 20) 5′-gTC.AgC.CAC.ACA.gTg.CCT.AT-3′ (located inGlycine max actin 1 gene (Accession J01298)) SOY02: (SEQ ID No.: 21)5′-gTT.ACC.gTA.CAg.gTC.TTT.CC-3′ (located in Glycine max actin 1 gene(Accession J01298))

2.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

For primer pair SOY01-SOY02: 413 bp (endogenous control) For primer pairDPA021-DPA024: 185 bp (A2704-12 elite Event)

2.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

2.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.

2.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:    -   2.5 μl template DNA    -   2.5 μl 10× Amplification Buffer (supplied with Taq polymerase)    -   0.5 μl 10 mM dNTP's    -   0.5 μl DPA021 (10 pmoles/μl)    -   0.5 μl DPA024 (10 pmoles/μl)    -   0.25 μl SOY01 (10 pmoles/μl)    -   0.25 μl SOY02 (10 pmoles/μl)    -   0.1 μl Taq DNA polymerase (5 units/μl)    -   water up to 25 μl

the thermocycling profile to be followed for optimal results is thefollowing:

-   -   4 min. at 95° C.    -   Followed by: 1 min. at 95° C.    -   1 min. at 57° C.    -   2 min. at 72° C.    -   For 5 cycles    -   Followed by: 30 sec. at 92° C.    -   30 sec. at 57° C.    -   1 min. at 72° C.    -   For 25 cycles    -   Followed by: 5 minutes at 72° C.

2.6. Agarose Gel Analysis

To optimally visualise the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder PHARMACIA).

2.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for A2704-12 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe A2704-12 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

2.8. Use of Discriminating PCR Protocol to Identify A2704-12

Before attempting to screen unknowns, a test run, with all appropriatecontrols, has to be performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Glycine max leaf material from a number of plants, some of whichcomprising A2704-12 were tested according to the above-describedprotocol. Samples from elite event A2704-12 and from Glycine maxwild-type were taken as positive and negative controls, respectively.

FIG. 2 illustrates the result obtained with the elite event PCRidentification protocol for A2704-12 on a number of soybean plantsamples (lanes 1 to 14). The samples in lane 1 were found to contain theelite event as the 185 bp band is detected, while the samples in lanes2, 3 and 4 do not comprise A2704-12. Lane 2 comprises another soybeanelite event, lane 3 represents a non-transgenic Glycine max control;lane 4 represents the negative control (water) sample, and lane 5represents the Molecular Weight Marker (100 bp).

3. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising A2704-12

A specific integration fragment of A2704-12 is obtained by PCRamplification using specific primers DPA021 (SEQ ID No. 4) and DPA024(SEQ ID No. 8) or by chemical synthesis and is labeled. This integrationfragment is used as a specific probe for the detection of A2704-12 inbiological samples. Nucleic acid is extracted from the samples accordingto standard procedures. This nucleic acid is then contacted with thespecific probe under hybridization conditions which are optimized toallow formation of a hybrid. The formation of the hybrid is thendetected to indicate the presence of A2704-12 nucleic acid in thesample. Optionally, the nucleic acid in the samples is amplified usingthe specific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

1. A method for identifying elite event A2704-12 in biological samples, comprising detecting a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12.
 2. The method of claim 1, said method comprising amplifying a DNA fragment of between 100 and 500 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ flanking region of A2704-12, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or the 3′ flanking region of A2704-12, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 3. The method of claim 2, wherein said primer recognizing the 5′ flanking region consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 4. The method of claim 2, wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 5. The method of claim 4, wherein said primer recognizing the 5′ flanking region comprises the sequence of SEQ ID No. 4 and said primer recognizing a sequence within the foreign DNA comprises SEQ ID No.
 8. 6. The method of claim 5, which method comprises amplifying a fragment of about 185 bp using the A2704-12 identification protocol.
 7. A kit for identifying elite event A2704-12 in biological samples, said kit comprising one primer recognizing the 5′ flanking region of A2704-12, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or one primer recognizing the 3′ flanking region of A2704-12, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and one primer recognizing a sequence within the foreign DNA, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 8. The kit of claim 7, wherein said primer recognizing the 5′ flanking region consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 9. The kit of claim 7, wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or said primer recognizing the 3′ flanking region of A2704-12 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide 1000, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 210 to nucleotide 720 or the nucleotide sequence of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 10. The kit of claim 7, comprising a primer consisting of the sequence of SEQ ID No. 4 and a primer consisting of the sequence of SEQ ID No.
 8. 11. A primer for use in a A2704-12 PCR identification protocol, comprising a sequence which, under optimized PCR conditions specifically recognizes a sequence within the 5′ or 3′ flanking region of A2704-12, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 and said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 12. The primer of claim 11, wherein said primer consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 13. The primer of claim 11, wherein said primer comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 209 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 569 to nucleotide
 1000. 14. A primer comprising at its extreme 3′ end the sequence of SEQ ID No.
 4. 15. A primer comprising at its extreme 3′ end the sequence of SEQ ID No.
 8. 16. The method of claim 1, which method comprises hybridizing a nucleic acid of biological samples with a specific probe for A2704-12.
 17. The method of claim 16, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith.
 18. The method of claim 17, wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences.
 19. A kit for identifying elite event A2704-12 in biological samples, said kit comprising a specific probe, capable of hybridizing specifically to a specific region of A2704-12.
 20. The kit of claim 19, wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith.
 21. The kit of claim 20, wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences.
 22. A specific probe for the identification of elite event A2704-12 in biological samples.
 23. The probe of claim 22, which has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of A2704-12 and the sequence of the foreign DNA contiguous therewith, or the complement thereof.
 24. The probe of claim 23 which has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences.
 25. A specific probe for the identification of elite event A2704-12 in biological samples, the sequence of being essentially similar to SEQ ID No. 1 from nucleotide 160 to 260 or SEQ ID No. 2 from nucleotide 520 to 620, or the complement of said sequences.
 26. A method for confirming seed purity, comprising detecting a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12, in seed samples.
 27. A method for screening seeds for the presence of A2704-12, comprising detecting a A2704-12 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of A2704-12, in samples of seed lots.
 28. A soybean plant, or cells, parts, seed or progeny thereof, comprising elite event A2704-12 in its genome. 